Rice-origin gibberellin 3beta-hydroxylase genes and utilization thereof

ABSTRACT

Genomic DNA and cDNA encoding GA 3β-hydroxylase were isolated from rice. When the expression of these genes was suppressed in rice plants, the plants became dwarfed compared with the wild type plants.

TECHNICAL FIELD

[0001] The present invention relates to rice genes involved ingibberellin biosynthesis and uses of these genes.

BACKGROUND ART

[0002] Multicellular organisms have a large number of specialized organsand tissues that are assembled to form a functional unit. Coordinationof various parts of an organism is achieved by chemical messengersubstances termed hormone. Plant hormones are naturally occurringsubstances, effective in very small amounts that act as signals tostimulate or inhibit growth or regulate development. Nowadays, thefollowing molecules are generally recognized to be plant hormones:auxins, gibberellins, cytokinins, abscisic acid, brassinolide, andethylene.

[0003] In animals, hormones are usually synthesized in special glandsand distributed via the bloodstream within the organism. Thus, theyreach the target site and responsive tissues that are ready to react.There they trigger specific regulatory processes. This classical conceptof hormones that was originally developed for animals was extended tohigher plants. In many cases, plant hormones are active in specifictarget tissues, which are often different from the tissues in which thehormone is produced. However, all the plant hormones can also bedetected in many tissues of the multicellular plant. This indicates thatthere is frequently no obligatory division between the site of synthesisand the site of action of plant hormones. If required, they are able toact on the same cells (tissues) in which they were synthesized. Thus,understanding the control of plant hormone synthesis is important indetermining the relationship between synthesis and action.

[0004] Gibberellins (GAs) were originally discovered as phytotoxins in1920s by Japanese phytopathologists. The pathogenic fungus, Gibberellafujikuroi, infects rice plants and secretes a compound that causespathological longitudinal growth (Bakanae “mad seedling disease”).Between 1935 and 1938 the active substance was isolated andcrystallized. It was called “gibberellin.” Later researches showed thatGAs are also synthesized by higher plants and are very important in theregulation of growth and in differentiation processes.

[0005] The basic structure of approximately 80 GAs identified up to 1992is the tetracyclic ring system of the ent-gibberellan (FIG. 1a). GAscontain diterpenoid carboxylic acids produced mainly from mevalonic acidvia cyclization of geranylgeranyl pyrophosphate (FIG. 1b). Most of theGAs are inactive in promoting plant development. In many plants,biologically active GAs, which act as plant growth regulators, are GA₁and GA₄. They can control various developmental processes, includingseed germination, stem elongation, flowering, and fruit development.Thus, various modified plants that are industrially useful can begenerated by modifying GA biosynthesis.

[0006] The role of GAs as mediators of environmental stimuli has beenwell established. Physical factors, such as light and temperature, canmodify GA metabolism by changing the flux through specific step in thepathway. For example, light quality (red or far-red) and intensity (highor low) affects GA biosynthesis. In lettuce seeds and cowpea epicotyls,3-beta hydroxylation of GA₂₀ is enhanced by treatment with far-red light(Toyumasu et al., (1992) Plant Cell Physiol. 33, 695-701). In addition,when pea seedlings are grown in low irradiance (40 μmol/m²s), GA₂₀content increases sevenfold compared with plants grown in highirradiance (386 μmol/m²s), whereas in plants grown in the dark, the GA₂₀content is reduced as compared to that in high irradiance (Gawronska etal., (1995) Plant Cell Physiol. 36, 1361-1367).

[0007] In spite of many attempts to implicate GA metabolism inphytochrome-mediated or light intensity-mediated changes in growth rate,supporting evidence is sparse. The mechanism(s) underlying theseregulatory processes will eventually be understood as a result of thecurrent advances in the molecular biology of GA biosynthesis.

[0008] Despite considerable efforts, the site of synthesis of bioactiveGAs and their mode of action in specific cells and tissues has not beenclarified. Based on experiments in which plants were supplemented with¹⁴C-labelled GAs, it is thought that they are translocated in anon-polar manner throughout the plant. More recently, graftingexperiments with dwarf and wild-type pea plants indicated that GA₁, oneof the bioactive GA, is not transported, unlike its precursor, GA₂₀,(Proebsting et al. (1992) Plant Physiol. 100, 1354-1360; Reid et al.,(1983) J. Exp. Bot. 34, 349-364). Quantitative analyses using GC-MS andbioassays with dwarf pea plants have revealed that GAs are mainlypresent in actively growing and elongating tissues such as shoot apices,young leaves, and flowers (Jones and Phillips, (1966) Plant Physiol. 41,1381-1386; Potts et al. (1982) Physiol. Plant, 55, 323-328: Kobayashi etal., (1988) Agric. Biol. Chem. 52, 1189-1194). However the exact amountof each GA in a specific tissue is difficult to determine because mostGAs are present in very small amounts and most are not bioactive.Therefore, a new approach is needed to clarify the location of synthesisof bioactive GAs.

[0009] According to progress in molecular biology and geneticengineering, almost of the genes encoding GA biosynthetic enzymes so farhave been cloned from various plant species. The studies of these cloneshave shown that GA responsive dwarf mutants lack the respective GAbiosynthetic enzymes (FIG. 1b). Their expression profile indicates thatthe pathway is strictly regulated during development. Among these genes,GA1 from Arabidopsis, which encodes copalyl diphosphate synthase (CPS),an enzyme active early in GA biosynthesis, is highly expressed inrapidly growing tissues, e.g., the shoot apex, root tips, and flowers(Silverstone et al., (1997) Plant J. 12, 9-19). GA C-20 oxidase, whichcatalyzes a late step in the GA biosynthetic pathway and constitutes asmall gene family, is specifically expressed in stems and developingseeds of Arabidopsis, pea, and bean where GA is required fordevelopment. They are negatively regulated by treatment with GA₃(Phillips et al., (1995) Plant Physiol. 108, 1049-1057; Garcia-Martinezet al., (1997) Plant Mol. Biol. 33, 1073-1084).

[0010] These observations lead the present inventors to speculate thatGA action in various organs may depend upon the amount of endogenous GApresent, which in turn depends on the regulation of expression of GAbiosynthetic enzymes, rather than on the translocation of bioactive GAto the site of GA action. However, analysis of the expression of CPS orGA C-20 oxidase provides no direct evidence of the site of synthesis ofbioactive GAs and regulation of bioactive GA levels because bioactiveGAs are synthesized by 3β-hydroxylation which is catalyzed by GA3β-hydroxylase.

[0011] As described above, 3β-hydroxylase catalyzes the conversion ofthe GA₂₀ and GA₉ to GA₁ and GA₄, respectively, at the final step in thesynthesis of bioactive GAs (FIG. 1b). The enzymology of 3β-hydroxylasehas not yet been completely clarified. However, the2-oxoglutarate-binding region is essential for its activity, indicatingthat GA 3β-hydroxylase has the typical properties of a2-oxoglutarate-dependent dioxygenase. Certain GA 3β-hydroxylase may bemultifunctional; the enzyme from pumpkin endosperm catalyzes both 2β and3β hydroxylation (Lange et al., (1997) Plant Cell, 9, 1459-1467). Maizedwarf-1,3β-hydroxylase has also been considered to be multifunctionaland it catalyzes three hydroxylation steps in the maize GA biosyntheticpathway (Spray et al., (1996) Proc. Natl. Acad. Sci. 93, 10515-10518).But the nature of these GA 3β-hydroxylases is still not well known.

DISCLOSURE OF THE INVENTION

[0012] The present invention provides novel GA 3β-hydroxylase genes fromrice and uses of the genes, especially for production of plants whoseplant type has been modified.

[0013] For the isolation of GA 3β-hydroxylase genes from rice, thepresent inventors first performed PCR using degenerate primers designedbased on the conserved region of dicot GA 3β-hydroxylases with the ricegenomic DNA as a template. Then, using a fragment of the genomic DNAencoding GA 3β-hydroxylase thus obtained as a probe, the presentinventors screened the rice genomic library to obtain several clones.These clones were divided into two groups based on their restrictionmaps, and one clone of each group was entirely sequenced. As a result,the present inventors found out that each of these clones encodes onerice GA 3β-hydroxylase.

[0014] Then, to obtain cDNA fragments based on the nucleotide sequenceof each clone, the present inventors performed RT-PCR using the totalRNA isolated from the rice seedlings (rice shoot apices) or unopenedflowers, thereby obtaining the full-length cDNA clones encoding GA3β-hydroxylases (designated “Os3β-1” and “Os3β-2”, respectively).Furthermore, using primers designed based on the genomic DNA sequencesthus obtained, the present inventors performed RT-PCR with the total RNAisolated from the rice seedlings (shoot apices) or unopened flowers as atemplate and succeeded in obtaining the cDNA clones encoding thecomplete rice GA 3β-hydroxylase.

[0015] Since in rice the d18 mutant is known as a GA-responsive dwarfcultivar, the present inventors examined whether the rice GA3β-hydroxylase clone thus isolated corresponds to the D18 gene. RFLP(Restriction Fragment Length Polymorphism) analysis and direct analysisof the nucleotide sequence of d18 allele proved that, of the twoisolated rice genes, the Os3β-2 gene is the causative gene of the d18mutation. In addition, the difference between the Os3β-1 gene and theOs3β-2 gene in their expression plant parts indicated that the Os3β-1protein is involved in a different biosynthetic pathway of bioactive GAsthan the Os3β-2 protein.

[0016] Furthermore, the present inventors succeeded in producing adwarfed rice plant compared with a wild type plant by suppressing theexpression of the Os3β-2 gene in rice plant utilizing the antisense DNAof the gene.

[0017] As described above, the present inventors succeeded in isolatinga novel GA 3β-hydroxylase gene from rice, and found that a plant withmodified plant type compared with a wild type plant can be produced bysuppressing the expression of the gene.

[0018] In more detail, the present invention is to provide:

[0019] (1) a DNA encoding a protein having the gibberellin3β-hydroxylase activity according to any one of the following (a)through (c):

[0020] (a) a DNA encoding a protein comprising the amino acid sequenceset forth in SEQ ID NO: 1 or 2,

[0021] (b) a DNA comprising the coding region of the nucleotide sequenceset forth in SEQ ID NO: 3 or 4.

[0022] (c) a DNA encoding a protein comprising the amino acid sequenceset forth in SEQ ID NO: 1 or 2 in which one or more amino acids aresubstituted, deleted, added, and/or inserted;

[0023] (2) a DNA encoding an antisense RNA complementary to the DNAaccording to (1) or its transcription product;

[0024] (3) a DNA encoding an RNA having the ribozyme activity tospecifically cleave the transcription product of the DNA according to(1);

[0025] (4) a DNA encoding an RNA that suppresses the expression ofendogenous DNA according to (1) by co-suppression when the endogenousDNA is expressed in plant cells;

[0026] (5) a vector containing the DNA according to any one of (1)through (4);

[0027] (6) a transformed plant cell harboring the DNA according to anyone of (1) through (4) in an expressible state;

[0028] (7) a transgenic plant containing the transformed plant cellaccording to (6);

[0029] (8) a propagative material of the transgenic plant according to(7);

[0030] (9) a protein encoded by the DNA according to (1);

[0031] (10) a method for producing the protein according to (9), whereinsaid method comprises culturing the transformed cells carrying the DNAaccording to (1) in an expressible state, and recovering the expressedprotein from said cells or the culture supernatant thereof;

[0032] (11) a method for modifying the plant growth, wherein said methodcomprises controlling the expression level of the DNA according to (1)in plant cells; and

[0033] (12) a method for modifying a plant type, wherein said methodcomprises controlling the expression level of the DNA according to (1)in plant cells.

[0034] The present invention provides novel GA 3β-hydroxylases isolatedfrom rice plant and DNAs encoding these enzymes. Nucleotide sequences ofcDNAs GA 3β-hydroxylase genes from rice, Os3β-1 and Os3β-2, isolated bythe present inventors and included in the DNA of this invention, are setforth in SEQ ID NOs: 3 and 4, respectively. The nucleotide sequence ofthe genomic DNAs of these cDNAs are shown in SEQ ID NOs: 5 and 6,respectively. In addition, amino acid sequences of the “Os3β-1” and“Os3β-2” proteins are set forth in SEQ ID NOs: 1 and 2, respectively.

[0035] Consistent with the previous classification of the 3β-hydroxylaseas 2-oxoglutarate-dependent dioxygenases (2-ODDs), both “Os3β-1 ” and“Os3β-2” proteins originating in rice contained all of the domainscharacteristic of plant 2-ODDs (Prescott, A. G. (1993) J. Exp. Bot. 44,849-861; de Carolis and Luca (1994) Phytochemistry 36, 1093-1107).Amongst all of the published sequences, coding regions of both clonesshow the highest homology with GA 3β-hydroxylases. Especially, theseregions are highly conserved (position of 240 to 247 and 302 to 307 inOs3β-1, 222 to 229 and 285 to 290 in Os3β-2) and may act as the bindingsite of iron and the cofactor, 2-oxoglutarate. These proteins also havethe conserved motif (Met-Trp-X-Glu-Gly-X-Thr), which is unique to the GA3β-hydroxylase (position of 144 to 150 in Os3β-1, 127 to 133 in Os3β-2).Sequence comparison with dicot GA 3β-hydroxylases and other dioxygenasessuggests that both cDNA clones isolated by the present inventors encoderice GA 3β-hydroxylases.

[0036] Mapping of Os3β-2 and genomic Southern analysis indicated thatOs3β-2 corresponds to the D18 gene. The rice d18 mutants areGA-responsive dwarf cultivars, including Hosetu-waisei, Akibare-waisei,Kotake-tamanishiki, and Waito-C (FIG. 2). Many dwarf alleles so far havebeen identified. The Os3β-2 protein is assumed to be also involved inthe internodal growth of plants via synthesis of bioactive GAs.

[0037] Analyses on the levels of endogenous GAs in different organs atvarious growth stages have revealed that 13-hydroxylated gibberellins(GA₁₉, GA₂₀, GA₁) are dominant in vegetative organs, whilenon-13-hydroxylated gibberellins (GA₂₄, GA₉, GA₄) are specificallyaccumulated in reproductive growth organs, especially in anthers. Thisindicates that the biosynthetic pathway for bioactive GAs isorgan-specific (Kurogouchi, S. et al. (1979) Planta 146, 185-191;Kobayashi, M. et al. (1984) Agric. Biol. Chem. 48, 2725-2729; Kobayashi,M. et al, (1988) Agric. Biol. Chem. 52, 1189-1194). Expression patternsof Os3β-2 (D18) and Os3β-1 are consistent with this speculation. Indeed,the Os3β-2 mRNA was in high level in stems, young leaves, andinflorescence meristems, while the Os3β-1 mRNA was specifically observedin flowers. These consistencies indicate that products of Os3β-2 andOs3β-1 may have the substrate-specificity for GA₂₀ and GA₉,respectively.

[0038] Expressions of Os3β-2 and Os3β-1 are also consistent with thedistribution of bioactive GAs in rice. Breeding and quantitativeanalyses revealed that GA₁ was high in young leaf tissues which are themost active sites of gibberellin biosynthesis (Choi, Y. -H. et al.(1995) Plant Cell Physiol. 36(6), 997-1001), and the highest expressionof Os3β-2 was also observed in young leaves. Interestingly, theexpression of Os3β-2 in shoot apices was at moderate levels as comparedwith other organs of rice, nevertheless many genes for GA biosynthesis,such as GA1 from Arabidopsis and Nty from tobacco, are stronglyexpressed in the actively dividing and elongating tissues, like shootapices and roots (Silverstone et al. (1997) Plant J. 12, 9-19). Thisdiscrepancy suggests that the organ activity to synthesize GAs maydiffer between monocotyledonous and dicotyledonous plants. On the otherhand, the endogenous level of GA₄ is extremely high in anthers at theflowering stage (Kobayashi, M. et al. (1988) Agric. Biol. Chem. 52,1189-1194; Kobayashi, M. et al. (1990) Plant Cell Physiol. 31(2),289-293). This fact is consistent with the specific expression of Os3β-1in flowers. This consistency may indicate that GA₄ is synthesized inanthers by the action of the Os3β-1 protein. Therefore, the Os3β-2 andOs3β-1 proteins may be involved in different biosynthetic pathways ofbioactive GAs.

[0039] In fact, the Os3β-1 protein catalyzed the production of GA₄(3β-hydroxylation), GA₇ (2,3-unsaturation and 3β-hydroxylation), andGA₃₄ (2β-hydroxylation) with GA₉ as its substrate (FIG. 9), and similarresults were obtained with GA₂₀ as the substrate. In addition, with GA₅and GA₄₄ as its substrate, the protein produced the corresponding3β-hydroxylated gibberellins, GA₃ and GA₈₈, respectively. Furthermore,the Os3β-2 protein catalyzed, with GA₅, GA₉, GA₂₀, and GA₄₄ as thesubstrates, the production of the corresponding 3β-hydroxylatedgibberellins, GA₃, GA₄, GA₁, and GA₃₈, respectively (FIG. 10) (Example5).

[0040] Several reports mention the necessity of bioactive GAs not onlyfor the stem elongation but also for a variety of other plant growthprocesses. For example, as to the floral organs, the male-sterilephenotype shown in an Arabidopsis GA-deficient mutant, gal-3 (Koornneef,M. and Van der Veen, J. H. (1980) Theor. Appl. Genet. 58, 257-263), andthe arrest of anther growth at an early stage without formation ofviable pollen grains in tomato mutants, stamenless-2 and gib-1 (Sawhney(1974) J. Exp. Bot. 25, 1004-1009; Jacobsen and Olszewski (1996) Proc.Natl. Acad. Sci. U.S.A. 93, 9292-9296), have been reported.

[0041] Since the proteins of the present invention are thought to beinvolved in the biosynthesis of bioactive GAs, they can be used inmanufacturing bioactive GAs. Furthermore, as described below, the plantgrowth can be modified by regulating expression levels of these proteinsin plants. For example, a plant having a different plant type from awild type can be produced.

[0042] The protein of this invention can be prepared as a recombinantprotein via methods known to those skilled in the art utilizing the generecombination techniques or as a natural protein. A recombinant proteincan be prepared, as described below, for example, by inserting DNA(e.g., SEQ ID NOs: 3 and 4) encoding the protein of this invention intoan appropriate expression vector and purifying the protein from cellstransformed with the vector. A natural protein can be prepared, forexample, by immunizing suitable animals with the prepared recombinantprotein or its partial peptide, binding the thus prepared antibody to acolumn for affinity chromatography, contacting the column with extractsprepared from tissues of tobacco and rice expressing the protein of thisinvention, and purifying the protein binding to the column.

[0043] The protein of this invention includes wild type proteins (SEQ IDNOs: 1 and 2) in which partial amino acid residues are modified, whileretaining the function of the wild type proteins. An example of themethod for preparing such modified proteins well known to those skilledin the art include the site-directed mutagenesis method (Kramer, W. &Fritz, H. -J. Oligonucleotide-directed construction of mutagenesis viagapped duplex DNA. Methods in Enzymology, 154: 350-367, 1987). Aminoacid mutations may also occur spontaneously. The protein of thisinvention thus include proteins that retain the GA 3β-hydroxylaseactivity of the wild-type protein and those that are modified viasubstitution, deletion, addition, and/or insertion of one or more aminoacid residues in the amino acid sequence of the wild type protein. Thereis no particular limitation on the site and number of such amino acidmodifications in the protein so far as the modified protein retains theGA 3β-hydroxylase activity. The number of amino acid that can bemodified is usually not more than 50 amino acid residues, preferably notmore than 30, more preferably not more than 10, and most preferably notmore than 3 amino acid residues.

[0044] Herein, the term “GA 3β-hydroxylase activity” refers to theactivity to synthesize GA₁ or GA₄ as the reaction product when GA₂₀ orGA₉ is used as a reaction substrate and ferrous iron and 2-oxoglutarateare used as cofactors. The activity can be detected, for example, asfollows. In general, cDNA obtained is inserted into an expression vectorand overexpressed as a fusion protein in E. coli. Using the cell extractthus obtained (as an enzyme solution), the reaction is performed invitro with GA₂₀ or G₉ as a reaction substrate in the presence of theco-factors, ferrous ion and 2-oxoglutarate, and finally the reactionproduct (GA₁ or GA₄) is confirmed by GC-MS.

[0045] The present inventors isolated cDNA and genomic DNA encoding theabove proteins. Therefore, DNAs encoding the protein of this inventioninclude both cDNA and genomic DNA so far as they encode these proteins.When DNAs encoding the Os3β-2 and Os3β-1 proteins are cDNAs, the DNAscan be prepared by RT-PCR using respective primers designed based on theinformation of nucleotide sequences set forth in SEQ ID NOs: 3 and 4 andthe total RNA isolated from seedlings (shoot apices of rice) or unopenedflowers as a template. Furthermore, the genomic DNA can be prepared byPCR using respective primers designed based on the information ofnucleotide sequences set forth in SEQ ID NOs: 5 and 6 and the ricegenomic DNA as a template.

[0046] DNAs encoding the protein of the present invention can be used,for example, for producing recombinant proteins. Production ofrecombinant proteins can be carried out described below. First, afull-length cDNA is synthesized by RT-PCR using primers provided withrestriction enzyme sites and subcloned into multi-cloning sites of thepMAL-c2 expression vector (NEB). This construct is used to transformEscherichia coli strain BL21 cells (protease-deficient strain) bystandard methods. Using the transformant thus obtained, the protein isinduced. E. coli are cultured (by shaking) in a 2×YT medium containing0.2% glucose at 37° C. When an OD₆₀₀ value reaches around 0.6, IPTG isadded to a final concentration of 1 mM, and culturing is furthercontinued at 18° C. for 24 h. Extraction of an enzyme solution isperformed as follows. After culturing, cells are collected and lysed ina suspension buffer (50 mM Tris-HCl (pH 8.0) containing 10% glycerol, 2mM DTT, and 1 mg/ml lysozyme). The cell suspension is allowed to standat 4° C. for 30 min, and then incubated at −80° C. until it becomescompletely frozen. The frozen suspension is thawed and sonicated for 30s twice at 5-min intervals at the MAX level with the Sonicator (HeatSystems—Ultrasonics, Inc., Model W-225R). The suspension thus treated iscentrifuged (at 15,000 rpm and 4° C. for 20 min), and the supernatant isused as a crude enzyme solution.

[0047] Furthermore, preparation of the purified protein can be carriedout, by expressing the protein of this invention in E. coli (or thelike) as a fusion protein with the histidine tag, maltose-bindingprotein, or glutathione-S-transferase (GST), and subsequently purifyingthem on a nickel column, an amylose-column, or a GST-glutathione column,respectively. Then, after the purification, the above-described tags canbe cleaved off using limited proteases, such as, thrombin and factor Xaas required.

[0048] As described above, the genes isolated by the present inventorsare assumed to be involved in the plant growth through the production ofbioactive GAs. Therefore, plant growth may be controlled by regulatingthe expression of these genes. Since Os3β-2 in particular is thought tobe involved in the internodal growth of plants, this gene may beutilized in the control of plant stature. Control of plant statureprovides a variety of industrial advantages.

[0049] For example, the shortened stature caused by suppressing theexpression of the gene of this invention in a plant can make the plantresistant to bending thereby increasing the fruit weight. Furthermore,the shortened stature makes the size of the plant per stub more compactso that the number of plants to be planted per unit area can beincreased. This dense planting is highly important in the production ofagricultural products including rice, wheat, maize, etc., in particular.DNA encoding the protein of the present invention may be applicable todwarf flowering plants, dwarf fruit trees, etc. Male sterile traits maybe induced by suppressing the expression of the Os3β-1 gene in flowers.

[0050] On the other hand, the yield of plants as a whole may be enhancedby lengthening plant stature through the elevated expression of genes ofthis invention within the plants. This is useful for improving feed cropyields as a whole in particular.

[0051] In the present invention, a variety of methods known to thoseskilled in the art are available for suppressing the expression of genesof this invention to control plant growth. Herein, “suppression ofexpression of genes” includes suppressions of both gene transcriptionand translation into proteins, and includes not only completesuppression but also decrease in the gene expression.

[0052] The expression of a specific endogenous gene in plants can besuppressed by conventional methods utilizing antisense technology. Eckeret al. were the first to demonstrate the effect of an antisense RNAintroduced by electroporation in plant cells by using the transient geneexpression method (Ecker, J. R. and Davis, R. W. (1986). Proc. Natl.Acad. Sci. USA 83, 5372). Thereafter, the target gene expression wasreportedly reduced in tobacco and petunias by expressing antisense RNAs(van der Krol, A. R. et al. (1988). Nature 333, 866). The antisensetechnique has now been established as a means to suppress target geneexpression in plants.

[0053] Multiple factors cause antisense nucleic acid to suppress thetarget gene expression. These include inhibition of transcriptioninitiation by triple strand formation; suppression of transcription byhybrid formation at the site where the RNA polymerase has formed a localopen loop structure; transcription inhibition by hybridization with theRNA being synthesized; suppression of splicing by hybrid formation atthe junction between an intron and an exon; suppression of splicing byhybrid formation at the site of spliceosome formation; suppression ofmRNA translocation from the nucleus to the cytoplasm by hybridizationwith mRNA; suppression of splicing by hybrid formation at the cappingsite or at the poly A addition site; suppression of translationinitiation by hybrid formation at the binding site for the translationinitiation factors; suppression of translation by hybrid formation atthe site for ribosome binding near the initiation codon; inhibition ofpeptide chain elongation by hybrid formation in the translated region orat the polysome binding sites of mRNA; and suppression of geneexpression by hybrid formation at the sites of interaction betweennucleic acids and proteins. These factors suppress the target geneexpression by inhibiting the process of transcription, splicing, ortranslation (Hirashima and Inoue, “Shin Seikagaku Jikken Koza (NewBiochemistry Experimentation Lectures) 2, Kakusan (Nucleic Acids) IV,Idenshi No Fukusei To Hatsugen (Replication and Expression of Genes),”Nihon Seikagakukai Hen (The Japanese Biochemical Society Ed.), TokyoKagaku Dozin, pp. 319-347, (1993)).

[0054] An antisense sequence used in the present invention can suppressthe target gene expression by any of the above-mentioned mechanisms. Ifan antisense sequence is designed to be complementary to theuntranslated region near the 5′ end of the gene's mRNA; it willeffectively inhibit translation of a gene. Additionally, it is alsopossible to use sequences that are complementary to the coding regionsor to the untranslated regions on the 3′ side. Thus, the antisense DNAused in the present invention includes a DNA having antisense sequencesagainst both the untranslated regions and the translated regions of thegene. The antisense DNA to be used is connected downstream from anappropriate promoter, and, preferably, a sequence containing thetranscription termination signal is connected on the 3′ side. The DNAthus prepared can be transfected into the desired plant by knownmethods. The sequence of the antisense DNA is preferably a sequencecomplementary to the endogenous gene (or the homologue) of the plant tobe transformed or a part thereof, but it need not be perfectlycomplementary so long as it can effectively inhibit the gene expression.The transcribed RNA is preferably not less than 90%, and most preferablynot less than 95% complementary to the transcribed products of thetarget gene. In order to effectively inhibit the expression of thetarget gene by means of an antisense sequence, the antisense DNA shouldbe at least 15 nucleotides long or more, preferably 100 nucleotides longor more, and most preferably 500 nucleotides long or more. The antisenseDNA to be used is generally shorter than 5 kb, and preferably shorterthan 2.5 kb.

[0055] DNA encoding ribozymes can also be used to suppress theexpression of endogenous genes. A ribozyme is defined as an RNA moleculethat has catalytic activities. Numerous ribozymes are known in theliterature, each having distinct catalytic activity. Research on theribozymes as RNA-cleaving enzymes has enabled the designing of aribozyme that site-specifically cleaves RNA. While some ribozymes of thegroup I intron type or the M1RNA contained in RNaseP consist of 400nucleotides or more, others belonging to the hammerhead type or thehairpin type have an activity domain of about 40 nucleotides (Koizumi,Makoto and Ohtsuka, Eiko (1990). Tanpakushitsu Kakusan Kohso (Protein,Nucleic acid, and Enzyme) 35, 2191).

[0056] The self-cleavage domain of a hammerhead type ribozyme cleaves atthe 3′ side of C15 sequence G13U14C15. Formation of a nucleotide pairbetween U14 and A at the ninth position is considered important for theribozyme activity. Furthermore, it has been shown that the cleavage alsooccurs when the nucleotide at the 15th position is A or U instead of C(Koizumi, M. et al. (1988). FEBS Lett. 228, 225). If thesubstrate-binding site of the ribozyme is designed to be complementaryto the RNA sequences adjacent to the target site, one can create arestriction-enzyme-like RNA cleaving ribozyme that recognizes thesequence UC, UU, or UA within the target RNA (Koizumi, M. et al. (1988).FEBS Lett. 239, 285; Koizumi, Makoto and Ohtsuka, Eiko (1990).Tanpakushitsu Kakusan Kohso (Protein, Nucleic acid, and Enzyme), 35,2191; Koizumi, M. et al. (1989). Nucleic Acids Res. 17, 7059). Forexample, in the coding region of the Nty gene, Os3β-1 gene, or Os3β-2gene (SEQ ID NO: 3 or 4) isolated by the present inventors, there arepluralities of sites that can be used as the ribozyme target.

[0057] The hairpin type ribozyme is also useful in the presentinvention. A hairpin type ribozyme can be found, for example, in theminus strand of the satellite RNA of tobacco ringspot virus (Buzayan, J.M. (1986). Nature 323, 349). This ribozyme has also been shown totarget-specifically cleave RNA (Kikuchi, Y. and Sasaki, N. (1992).Nucleic Acids Res. 19, 6751; Kikuchi, Yo (1992) Kagaku To Seibutsu(Chemistry and Biology) 30, 112).

[0058] The ribozyme designed to cleave the target is fused with apromoter, such as the cauliflower mosaic virus 35S promoter, and with atranscription termination sequence, so that it will be transcribed inplant cells. However, if extra sequences are added to the 5′ end or the3′ end of the transcribed RNA, the ribozyme activity may be lost. Inthis case, one can place an additional trimming ribozyme, whichfunctions in the cis position to perform the trimming on the 5′ or the3′ side of the ribozyme portion, thereby precisely cutting the ribozymeportion from the transcribed RNA containing the ribozyme (Tairas, K. etal. (1990). Protein Eng. 3, 733; Dzaianott, A. M. and Bujarski, J. J.(1989). Proc. Natl. Acad. Sci. USA 86, 4823; Grosshands, C. A. and Cech,R. T. (1991). Nucleic Acids Res. 19, 3875; Taira, K. et al. (1991.)Nucleic Acid Res. 19, 5125). Multiple sites within the target gene canbe cleaved by arranging these structural units in tandem to achievegreater effects (Yuyama, N. et al., (1992). Biochem. Biophys. Res.Commun. 186, 1271). By using such ribozymes, it is possible tospecifically cleave the transcription products of the target gene in thepresent invention, thereby suppressing the expression of the gene.

[0059] Endogenous gene expression can also be suppressed byco-suppression through the transformation by DNA having a sequenceidentical or similar to the target gene sequence. “Co-suppression,” asused herein, refers to the phenomenon in which, when a gene having asequence identical or similar to the target endogenous gene sequence isintroduced into plants by transformation, expression of both theintroduced exogenous gene and the target endogenous gene becomessuppressed. Although the detailed mechanism of co-suppression isunknown, it is frequently observed in plants (Curr. Biol. (1997). 7,R793, Curr. Biol. (1996). 6, 810). For example, if one wishes to obtaina plant body in which the gene of the present invention isco-suppressed, the plant in question can be transformed with a DNAvector designed so as to express the gene of the present invention orDNA having a similar sequence. The gene to be used for co-suppressionneed not be completely identical to the target gene. However, it shouldhave preferably 70% or more sequence identity, more preferably 80% ormore sequence identity, and most preferably 90% or more (e.g. 95% ormore) sequence identity.

[0060] The identity of one amino acid sequence or nucleotide sequence toanother can be determined by following the BLAST algorithm by Karlin andAltschl (Proc. Natl. Acad. Sci. USA, (1993). 90, 5873-5877,). Programssuch as BLASTN and BLASTX were developed based on this algorithm(Altschul et al. (1990). J. Mol. Biol.215, 403-410). To analyze anucleotide sequences according to BLASTN based on BLAST, the parametersare set, for example, as score=100 and word length=12. On the otherhand, parameters used for the analysis of amino acid sequences by theBLASTX based on BLAST include, for example, score=50 and word length=3.Default parameters of each program are used when using BLAST and GappedBLAST programs. Specific techniques for such analysis are known in theart (http://www.ncbi.nlm.nih.gov.)

[0061] Modification of plant growth utilizing a DNA functioning tosuppress the expression of genes of this invention may be achieved byinserting the DNA into an appropriate vector, transferring the vectorinto plant cells, and regenerating the transformed plant cells thusobtained. There is no particular limitation on the type of vectors sofar as they are capable of expressing the inserted gene within plantcells.

[0062] For example, the promoter for the gene isolated by the presentinventors may be used. A vector having a promoter (for example, 35Spromoter of cauliflower mosaic virus) that enables the constitutive geneexpression in plant cells may also be used. Furthermore, planttissue-specific promoters may specifically modify particular planttissues, for example, leaves, flowers, fruits, etc. Examples of thetissue-specific promoters are seed-specific promoters such as promotersfor β-phaseolin of kidney bean (Bustos, et al. (1991). EMBO J. 10,1469-1479) and glycinin of soy bean (Lelievre, et al. (1992). PlantPhysiol. 98, 387-391); leaf-specific promoters such as promoters for theRbcS gene of pea (Lam and Chua (1990). Science 248, 471-474) and Cab 1gene of wheat (Gotorn, et al. (1993). Plant J. 3, 509-518),root-specific promoters such as promoters for the TobRB7 gene of tobacco(Yamamoto, et al. (1991). Plant Cell 3, 371-382) and rolD gene ofAgrobacterium rhizogenes (Elmayan and Tepfer (1995). Transgenic Res. 4,388-396). It is also possible to use a vector having a promoterinducibly activated by exogenous stimuli.

[0063] Although there is no particular limitation on the type of plantcells into which a vector is inserted, rice and tobacco, from which thegenes of the present invention are derived, are particularly preferred.Herein, the term “plant cells” includes plant cells in a variety offorms, for example, cultured cell suspension, protoplasts, leafsections, cali, etc. A vector can be transferred into plant cells by avariety of methods well known to those skilled in the art, including thepolyethylene glycol method, electroporation method,Agrobacterium-mediated method, particle gun method, etc. Regeneration ofa plant body from transformed plant cells may be performed by thestandard methods known in the art. Once the transformed plant body isgenerated, it is possible to obtain propagative materials (for example,seeds, tubers, cuttings, etc.) from the plant body and produce thetransformed plant of this invention on a large scale.

[0064] Furthermore, in this invention, it may also be possible toenhance the plant growth by promoting the expression of DNA isolated bythe present inventors. In this case, the DNA is inserted into anappropriate vector, and the resulting recombinant vector is transferredinto plant cells so as to regenerate the transformed plant cells thusobtained. Vectors used for the expression in plant cells, plant cellsinto which vectors are transferred, methods for regenerating plantbodies are similar to those in the case where the above-describedantisense DNAs and the like are used.

BRIEF DESCRIPTION OF THE DRAWINGS

[0065]FIG. 1a shows a general structure of gibberellin(ent-gibberellan).

[0066]FIG. 1b shows a major GA biosynthetic pathway in higher plants.

[0067] The italicized letters indicate a GA-responsive dwarf mutant thatlacks a specific GA biosynthetic pathway: ga1, ga2, ga3, ga5, and ga4from Arabidopsis; an1, d5, d3, and d1 from maize; ls and le from pea;d35 (dx) and d18 (dy) from rice.

[0068]FIG. 2 is a photograph showing various phenotypes of d18 dwarfplants. From left to right, Taichuu-65 (WT: approximately 1 m at thefinal stage), Kotake-tamanishiki dwarf (d18^(k): approximately 65 cm),Waito-C (approximately 55 cm), Hosetsu-waisei dwart (d18^(h):approximately 15 cm), and Akibare-waisei dwart (Bar=10 cm).

[0069]FIG. 3 shows the loci of GA 3β-hydroxylase (Os3β-1 and Os3β-2)genes and various RFLP makers on rice chromosomes 1 and 5.

[0070]FIG. 4a is an electrophoretogram representing the results of RFLPanalysis of the D18 and d18 alleles. DNA was isolated from leaf tissuesof D18 (Shiokari and Akibare) and d18 allele (d18^(h), ld18^(h), andd18-AD) plants. The DNA was digested with ApaI, separated byelectrophoresis, bound to nylon filters, and then hybridized. Molecularlength markers are given at left in kilobases.

[0071]FIG. 4b is a restriction map of genomic Os3β-2 clone and itssubclone. Genomic clone was obtained by screening the genomic libraryusing the PCR product as a probe. The 2.3-kb BglII fragment subclonecontains the entire coding region of D18.

[0072]FIG. 5 is an electrophoretogram representing expression patternsof GA 3β-hydroxylases in wild type Oryza sativa plants. The patternswere obtained by hybridization of 3β-hydroxylase cDNA D18 and Os3β-1 tonorthern blots from 10 μg of total RNA extracted from SA (shoot apices),ST (stems), LB (leaf blades), Ra (rachises), FL (flowers), YL (youngleaves), IFM (inflorescence meristems), and Sh (2-week-old seedlings).

[0073]FIG. 6 is a photograph representing transformed plants in whichthe Os3β-2 (D18) cDNA is constitutively expressed in the antisenseorientation under the control of the actin promoter. A wild typenipponbare plant is on the left, a semi-dwarfed plant on the middle, anda dwarfed plant on the right.

[0074]FIG. 7 shows the plasmid pBS-SK⁺ into which the full-length Os3β-2cDNA has been inserted.

[0075]FIG. 8 shows the plasmid pAct-NOS/Hm2 into which the antisenseOs3β-2 (D18) gene has been inserted.

[0076]FIG. 9 shows the pathway of the production of GA₄, GA₇, and GA₃₄catalyzed by an Os3β-1 fusion protein when GA₉ is used as a substrate.

[0077]FIG. 10 shows the pathway of the production of 3β-hydroxylatedgibberellins (GA₃, GA₄, GA₁, and GA₃₈) corresponding to GA₅, GA₉, GA₂₀,and GA₄₄, respectively, which are used as substrates, catalyzed by anOs3β-2 fusion protein.

BEST MODE FOR CARRYING OUT THE INVENTION

[0078] The present invention will be explained in detail below withreference to Examples, but is not to be construed as being limitedthereto. Rice seeds (Oryza sativa, Japonica-type cultivars:“Nipponbare”, “Akibare”, “Shiokari” and others) were sterilized in 1%NaClO for 1 h and thoroughly rinsed in sterile distilled water andgerminated on soil and grown at a greenhouse.

EXAMPLE 1 Isolation of cDNA clone encoding GA 3β-hydroxylase

[0079] No report on isolation of GA 3β-hydroxylase in monocots isavailable. Several GA 3β-hydroxylases have been cloned from dicots(Chiang et al., 1995; Martin et al., 1997; Lester et al., 1997).

[0080] For the isolation of a partial fragment encoding GA3β-hydroxylase, PCR was performed using the rice genomic DNA as atemplate and degenerate primers (5′ primer:5′-GTNGTNAARGTNGGNGARRT-3′/SEQ ID NO: 7; 3′ primer:5′-AYYTARTCRTTGGANGTNAC-3′/SEQ ID NO: 8) designed from the conservedregion among the reported dicot's GA 3β-hydroxylase sequences. A 210-bpDNA fragment, which corresponds to the size expected from the reportedGA 3β-hydroxylase sequences, was obtained.

[0081] To isolate full-length clones, the present inventors screened therice genomic library with this fragment as a probe. Several clones wereisolated and were divided into two groups based on the restriction mapof each genomic clone. Finally one clone of each group was entirelysequenced and designated as Os3β-1 and Os3β-2 (Os3β-1 and Os3β-2; Orizasativa GA 3β-hydroxylase −1, −2). Nucleotide sequences of these cloneswere set forth in SEQ ID NOs: 5 and 6. Os3β-1 shared the sequence withthe fragment used as a probe, but Os3β-2 contained a different sequenceat the corresponding region from the fragment.

[0082] Based on the sequence of each clone, the present inventorsperformed RT-PCR using the total RNA isolated from seedlings (rice shootapices) or unopened flowers to obtain cDNA fragment. As a result,full-length cDNA clones encoding GA 3β-hydroxylases were obtained. EachcDNA, Os3β-1 or Os3β-2, contained an open reading frame encoding apolypeptide with 379 or 373 amino acids, respectively. Nucleotidesequences of these clones are set forth in SEQ ID NOs: 3 and 4. TheOs3β-2 genomic DNA contained a single short intron (110 bp) and theintron was located at the same position as previously reported GA3β-hydroxylases in dicot. The genomic DNA of the other clone, Os3β-1,contained two introns. One was located at the same position as theintron of Os3β-2 and its size was comparable to that of Os3β-2 (110 bp).The other was located at the position of the binding site of theco-factor, 2-oxoglutarate (400 bp) (data not shown). The deduced aminoacid sequences of both clones shared a high degree of similarity toother GA 3β-hydroxylases, and they showed the highest similarity to eachother (56.6% identity, and 88.2% similarity).

[0083] Nucleotide sequences were determined by the dideoxynucleotidechain-termination method using an automated sequencing system (ABI373A).Analysis of sequences was carried out using GENETYX computer software(Software Kaihatsu Co., Japan).

EXAMPLE 2 Identification and characterization of d18 alleles

[0084] Previous quantitative analyses and bioassay of the dwarf riceplants indicated that the D18 gene encodes a GA 3β-hydroxylase. The D18locus has been identified on chromosome 1, flanking to the FS-2 locus atthe bottom of this chromosome. To investigate that the isolated GA3β-hydroxylase clones correspond to the D18 gene, the present inventorsmapped two clones on the rice genome using RFLP (Restriction FragmentLength Polymorphism) analysis.

[0085] RFLP of Os3β-1 or Os3β-2 were present between Asominori (aJaponica rice) and IR 24 (an Indica rice) DNAs digested with EcoRI orApaI, respectively. Linkage analysis was performed with digested genomicDNA from F2 progeny of crosses between Asominori and IR 24. Os3β-1 andOs3β-2 are mapped on the top of chromosome 5 and the bottom ofchromosome 1, respectively (FIG. 3). This result suggests that theOs3β-2 locus corresponds to the D18 locus.

[0086] Further analysis was performed to confirm that Os3β-2 is locatedat the D18 locus. There are four independent conventional mutantsgenerated by the loss-of-function of the D18 gene. The mutations may becaused by the DNA rearrangements and/or deletions of the D18 gene bymutagenesis using γ-irradiation. Therefore, RFLP at the position ofOs3β-2 between a wild type and these mutants may be observed if Os3β-2is the D18 gene.

[0087] DNA gel blot analysis of genomic DNA from a wild type (Shiokarior Akibare) and the d18 alleles (ld18^(k), ld18^(h), and d18-AD) wasperformed. ld18^(k) and ld18^(h) are isogenic lines of Shiokaribackground, and d18-AD was isolated from an Akibare mutant generated bymutagenesis with ethylenimines (EI). For DNA gel blot analysis, ricegenomic DNA (1 μg per lane) was digested with restriction enzymes,separated by agarose gel electrophoresis, and transferred to Hybond N+nylon membrane (Amersham) (Sambrook et al., 1989). Hybridization wasperformed at 65° C. in 0.25 M Na₂HPO₄, 1 mM EDTA, and 7% SDS. Filterswere washed twice for 15 min at 65° C. in 2×SSC, 0.1% SDS and once in0.1×SSC, 0.1% SDS at 65° C. for 15 min.

[0088] Eight enzymes (BamHI, BglII, ApaI, KpnI, DraI, EcoRV, EcoRI, andHindIII) were used to digest these genomic DNA to find RFLP between thewild type plants and the mutants. Polymorphisms were observed when theDNAs from d18-AD and ld18^(h) were digested with ApaI, while ld18^(k)did not show any polymorphisms when digested with any enzymes tested(FIG. 4).

[0089] The result of RFLP analysis strongly suggests that d18-AD has along deletion in the D18 locus, while ld18^(h) has a short deletionincluding the ApaI site. To confirm the result, the present inventorsanalyzed the entire coding sequences of all d18 alleles and comparedthem with that of the wild type Os3β-2.

[0090] Specifically, oligonucleotide primers designed based on the 5′and 3′ noncoding sequences of D18 were used to amplify the 1.6-kbfragments containing the entire coding region from D18, d18^(h), d18k,and d18-w, and then the amplified fragments were sequenced.

[0091] As expected, the Os3β-2 coding sequence was altered in all d18alleles at various positions (Table 1), while the d18-AD produced no RCRproduct. TABLE 1 Position in Nature of coding Consequence Allelemutation^(a) sequence of mutation d18-AD 7-kb absence of (Akibare-deletion a full length waisei) of D18 ORF d18^(h) GGG to GG, Gly²⁵¹frameshift, (Hosetsu- 1-base addition of 38 waisei) deletion novel aminoacids, truncated polypeptide d18^(k) CGC to TGC Arg¹⁴⁵ amino acidsubstitution, (Kotake- substitute Asp to Cys, tamanishiki) alteredproduct d18-w 9-base deletion Val⁵⁷ to Arg⁵⁹ in-frame deletion,(Waito-C) absence of a 3-amino-acid residue segment altered product

[0092] This supports the above result that d18-AD almost entirely lostthe coding sequence (data not shown). In the sequence from ld18^(k),substitution of C to T at the nucleotide position 433 numbering from thestart codon, converted Arginine-168 to Cystine. In d18-w, in-framedeletion of 9 nucleotides at the 15 position of 169 to 177, resulted indeletion of 3 amino acids Valine-57 to Arginine-59. In ld18^(h),deletion of nucleotide G caused a reading frame shift. These resultsdemonstrate that the Os3β-2 gene is located at the D18 locus, andencodes an GA 3β-hydroxylase.

EXAMPLE 3 Regulation of the Os3β-2 (D18) and Os3β-1 genes during plantgrowth

[0093] To investigate the expression of the D18 and Os3β-1 genes duringplant growth, RNA gel blot analysis was performed. For this analysis,total RNA was prepared from various organs or tissues by the standardmethod (Sambrook et al., 1989). RNA (10 μg per sample) was separated bygel electrophoresis and transferred to a Hybond N+ nylon membrane(Amersham). Hybridization was performed at 65° C. in a solutioncontaining 5×SSC, 10% (w/v) dextran sulfate, 0.5% (w/v) SDS, 0.1 mg/mldenatured salmon sperm DNA. Filters were washed twice for 15 min at 65°C. in 2×SSC, 0.1% SDS and once in 0.1×SSC, 0.1% SDS at 65° C. for 15min. A BssHII-PvuII (519 bp) fragment from full-length D18 cDNA and aKpnI-PvuII (310 bp) fragment from Osβ-1, were used as probes.

[0094] D18 gene was expressed in every organ tested (FIG. 5). The levelsof expression were high in stems, young leaves, and inflorescencemeristems, and low in leaf blades and rachises. In contrast, the Os3β-1mRNA expression was specifically high in the flowers, and low in leafblades and rachises.

[0095] Because D18 and Os3β-1 are highly similar to each other, thepresent inventors examined the degree of cross-hybridization by genomicsouthern analysis. When the respective specific probe was used togenomic southern hybridization, cross-hybridization was not detected(data not shown).

EXAMPLE 4 Production of dwarfed plant by suppressing expression ofOs3β-2 (D18) gene

[0096] A full-length Os3β-2 cDNA that had been cloned to theBamHI-HindIII site in the pBS-SK+ plasmid (FIG. 7), was cleaved out fromthe vector by the BamHI-HindIII digestion to collect the cDNA, and thenits ends were blunted. This blunted full-length cDNA was inserted to theSmaI site of the pAct-NOS/Hm2 plasmid to construct a vector to expressthe antisense Os3β-2 gene (FIG. 8).

[0097] Agrobacterium strain EHA101 was transformed with this recombinantplasmid by the electroporation method. Germinating seeds of rice werecontacted with the Agrobacterium strain and cultured in a selectionmedium containing kanamycin and hygromycin for 3 weeks to selectresistant cells, which were transplanted to a re-differentiation mediumso as to obtain dozens of transgenic plants. As a result, plantsexpressing the antisense Os3β-2 gene became dwarfed compared with thewild type rice plant (FIG. 6).

EXAMPLE 5 Function of recombinant GA 3β-hydroxylase

[0098] cDNAs for the predicted protein-coding regions of the Os3β-1 andOs3β-2 genes were each inserted into the pMAL-c2 expression vector (NewEngland Biolabs, Beverly, Mass.) in the sense orientation to obtain afusion translation product. The constructs thus obtained, pMAL-Os3β-1and pMAL-Os3β-2, were expressed in E. coli strain JM109. Bacterial cellswere cultured by shaking in 2×YT medium containing 100 mg/L ampicillinat 30° C. overnight. Then, the culture was diluted 100-fold with a fresh2×YT medium containing 100 mg/L ampicillin, and further cultured withshaking at 30° C. Four hours later, IPTG was added to the culture to afinal concentration of 1 mM, and the mixture was incubated with shakingat 17° C. for further 18 h. After the completion of culturing, thebacterial cells were collected, washed with a washing buffer (containing50 mM Tris HCl (pH 8.0), 10% (w/v) glycerol, and 2 mM DTT), suspended inthe washing buffer containing lysozyme (1 mg/ml), and allowed to standfor 30 min on ice.

[0099] The cell lysate thus obtained was sonicated, centrifuged, and thesupernatant was subjected to SDS-PAGE to confirm the expression of thefusion protein. For the assay of enzymatic activity of the Os3β-1 fusionprotein, the supernatant was incubated with various gibberellins andcofactors (ascorbate, ferrous iron, and 2-ketoglutarate). For theactivity assay of the Os3β-2 fusion protein, the supernatant waspurified on a column of amylose resin according to the method describedin the manual, and the purified protein thus obtained was similarlyincubated as described above. Metabolized gibberellins were identifiedusing GC-MS. In the case of the Os3β-1 fusion protein, the synthesis ofGA₄ (3β-hydroxylation), GA₇ (2,3-unsaturation and 3β-hydroxylation), andGA₃₄ (2β-hydroxylation) were confirmed when the substrate was GA₉ (FIG.9), with GA₄ and GA₇ being dominant among these reaction products.Similar results were obtained when GA₂₀ was used as a substrate.Furthermore, when GA₅ and GA₄₄ were the substrate, only thecorresponding 3β-hydroxylated gibberellins (GA₃ and GA₈₈) were obtained.On the other hand, when GA₅, GA₉, GA₂₀, and GA₄₄ were used as thesubstrate, the Os3β-2 fusion protein produced the corresponding3β-hydroxylated gibberellins (GA₃, GA₄, GA₁, and GA₃₈) (FIG. 10).

[0100] The results revealed that the Os3β-1 gene encodes an enzymecatalyzing the nuclear reactions of 2-, 3-unsaturation and2β-hydroxylation as well as 3β-hydroxylation. Furthermore, it becameevident that the Os3β-2 gene encodes an enzyme catalyzing the3β-hydroxylation.

INDUSTRIAL APPLICABILITY

[0101] The present invention has provided novel proteins and genesinvolved in the activation of plant gibberellins as well as plants whosegibberellin activity has been modified by controlling the expression ofthese genes. This invention enables modification of gibberellinactivation in plants so as to artificially modify the plant types.Suppression of gibberellin activation in plants induces plant dwarfphenotypes due to suppression of longitudinal growth. For example, thiscould prevent rice plants from bending over when excessive elongation ispromoted by ample fertilization. A substantial increase in crops mayalso be expected due to enhanced efficiency of light reception toleaves. It is also possible to improve efficiency in harvesting andbreeding management. Another result of the present invention is toincrease the yield of the plant as a whole by elevating the expressionof genes of this invention in the plant so as to promote gibberellinactivation therein. This strategy is particularly beneficial inimproving the yield of feed crops as a whole.

1 8 1 379 PRT Oryza sativa 1 Met Thr Ser Ser Ser Thr Ser Pro Thr Ser ProLeu Ala Ala Ala Ala 1 5 10 15 His Asn Gly Val Thr Ala Ala Tyr Phe AsnPhe Arg Gly Ala Glu Arg 20 25 30 Val Pro Glu Ser His Val Trp Lys Gly MetHis Glu Lys Asp Thr Ala 35 40 45 Pro Val Ala Ala Ala Asp Ala Asp Gly GlyAsp Ala Val Pro Val Val 50 55 60 Asp Met Ser Gly Gly Asp Asp Ala Ala ValAla Ala Val Ala Arg Ala 65 70 75 80 Ala Glu Glu Trp Gly Gly Phe Leu LeuVal Gly His Gly Val Thr Ala 85 90 95 Glu Ala Leu Ala Arg Val Glu Ala GlnAla Ala Arg Leu Phe Ala Leu 100 105 110 Pro Ala Asp Asp Lys Ala Arg GlyAla Arg Arg Pro Gly Gly Gly Asn 115 120 125 Thr Gly Tyr Gly Val Pro ProTyr Leu Leu Arg Tyr Pro Lys Gln Met 130 135 140 Trp Ala Glu Gly Tyr ThrPhe Pro Pro Pro Ala Ile Arg Asp Glu Phe 145 150 155 160 Arg Arg Val TrpPro Asp Ala Gly Asp Asp Tyr His Arg Phe Cys Ser 165 170 175 Ala Met GluGlu Tyr Asp Ser Ser Met Arg Ala Leu Gly Glu Arg Leu 180 185 190 Leu AlaMet Phe Phe Lys Ala Leu Gly Leu Ala Gly Asn Asp Ala Pro 195 200 205 GlyGly Glu Thr Glu Arg Lys Ile Arg Glu Thr Leu Thr Ser Ser Thr 210 215 220Ile His Leu Asn Met Phe Pro Arg Cys Pro Asp Pro Asp Arg Val Val 225 230235 240 Gly Leu Ala Ala His Thr Asp Ser Gly Phe Phe Thr Phe Ile Leu Gln245 250 255 Ser Pro Val Pro Gly Leu Gln Leu Leu Arg His Arg Pro Asp ArgTrp 260 265 270 Val Thr Val Pro Gly Thr Pro Gly Ala Leu Ile Val Val ValGly Asp 275 280 285 Leu Phe His Val Leu Thr Asn Gly Arg Phe His Ser ValPhe His Arg 290 295 300 Ala Val Val Asn Arg Glu Arg Asp Arg Ile Ser MetPro Tyr Phe Leu 305 310 315 320 Gly Pro Pro Ala Asp Met Lys Val Thr ProLeu Val Ala Ala Gly Ser 325 330 335 Pro Glu Ser Lys Ala Val Tyr Gln AlaVal Thr Trp Pro Glu Tyr Met 340 345 350 Ala Val Arg Asp Lys Leu Phe GlyThr Asn Ile Ser Ala Leu Ser Met 355 360 365 Ile Arg Val Ala Lys Glu GluAsp Lys Glu Ser 370 375 2 373 PRT Oryza sativa 2 Met Pro Thr Pro Ser HisLeu Lys Asn Pro Leu Cys Phe Asp Phe Arg 1 5 10 15 Ala Ala Arg Arg ValPro Glu Thr His Ala Trp Pro Gly Leu Asp Asp 20 25 30 His Pro Val Val AspGly Gly Gly Gly Gly Gly Glu Asp Ala Val Pro 35 40 45 Val Val Asp Val ArgAla Gly Asp Ala Ala Ala Arg Val Ala Arg Ala 50 55 60 Ala Glu Gln Trp GlyAla Phe Leu Leu Val Gly His Gly Val Pro Ala 65 70 75 80 Ala Leu Leu SerArg Val Glu Glu Arg Val Ala Arg Val Phe Ser Leu 85 90 95 Pro Ala Ser GluLys Met Arg Ala Val Arg Gly Pro Gly Glu Pro Cys 100 105 110 Gly Tyr GlySer Pro Pro Ile Ser Ser Phe Phe Ser Lys Leu Met Trp 115 120 125 Ser GluGly Tyr Thr Phe Ser Pro Ser Ser Leu Arg Ser Glu Leu Arg 130 135 140 ArgLeu Trp Pro Lys Ser Gly Asp Asp Tyr Leu Leu Phe Cys Asp Val 145 150 155160 Met Glu Glu Phe His Lys Glu Met Arg Arg Leu Ala Asp Glu Leu Leu 165170 175 Arg Leu Phe Leu Arg Ala Leu Gly Leu Thr Gly Glu Glu Val Ala Gly180 185 190 Val Glu Ala Glu Arg Arg Ile Gly Glu Arg Met Thr Ala Thr ValHis 195 200 205 Leu Asn Trp Tyr Pro Arg Cys Pro Glu Pro Arg Arg Ala LeuGly Leu 210 215 220 Ile Ala His Thr Asp Ser Gly Phe Phe Thr Phe Val LeuGln Ser Leu 225 230 235 240 Val Pro Gly Leu Gln Leu Phe Arg Arg Gly ProAsp Arg Trp Val Ala 245 250 255 Val Pro Ala Val Ala Gly Ala Phe Val ValAsn Val Gly Asp Leu Phe 260 265 270 His Ile Leu Thr Asn Gly Arg Phe HisSer Val Tyr His Arg Ala Val 275 280 285 Val Asn Arg Asp Arg Asp Arg ValSer Leu Gly Tyr Phe Leu Gly Pro 290 295 300 Pro Pro Asp Ala Glu Val AlaPro Leu Pro Glu Ala Val Pro Ala Gly 305 310 315 320 Arg Ser Pro Ala TyrArg Ala Val Thr Trp Pro Glu Tyr Met Ala Val 325 330 335 Arg Lys Lys AlaPhe Ala Thr Gly Gly Ser Ala Leu Lys Met Val Ser 340 345 350 Thr Asp AlaAla Ala Ala Ala Asp Glu His Asp Asp Val Ala Ala Ala 355 360 365 Ala AspVal His Ala 370 3 1187 DNA Oryza sativa CDS (1)..(1137) 3 atg aca tcgtcg tcg acc tcg ccg acc tcg ccg ctg gcc gcc gcc gca 48 Met Thr Ser SerSer Thr Ser Pro Thr Ser Pro Leu Ala Ala Ala Ala 1 5 10 15 cac aat ggcgtc acc gcc gcc tac ttc aac ttc cgc ggg gcg gag cgc 96 His Asn Gly ValThr Ala Ala Tyr Phe Asn Phe Arg Gly Ala Glu Arg 20 25 30 gtg ccg gag tcgcac gtg tgg aag ggg atg cac gag aag gac acc gcg 144 Val Pro Glu Ser HisVal Trp Lys Gly Met His Glu Lys Asp Thr Ala 35 40 45 ccg gtg gcg gcg gcggac gcg gac ggc ggc gac gcg gtg ccg gtg gtg 192 Pro Val Ala Ala Ala AspAla Asp Gly Gly Asp Ala Val Pro Val Val 50 55 60 gac atg agc ggc ggc gacgac gcc gcg gtg gcg gcg gtg gcg cgc gcg 240 Asp Met Ser Gly Gly Asp AspAla Ala Val Ala Ala Val Ala Arg Ala 65 70 75 80 gcg gag gag tgg ggc gggttc ctg ctc gtc ggg cac ggc gtg acc gcg 288 Ala Glu Glu Trp Gly Gly PheLeu Leu Val Gly His Gly Val Thr Ala 85 90 95 gag gcc ctg gcg cgc gtc gaggcg cag gcg gcg cgg ctg ttc gcg ctg 336 Glu Ala Leu Ala Arg Val Glu AlaGln Ala Ala Arg Leu Phe Ala Leu 100 105 110 ccg gcg gac gac aag gcg cgcggg gcg cgg cgg ccc ggc ggc ggg aac 384 Pro Ala Asp Asp Lys Ala Arg GlyAla Arg Arg Pro Gly Gly Gly Asn 115 120 125 acc ggc tac ggc gtg ccg ccgtac ctc ctc cgg tac ccg aag cag atg 432 Thr Gly Tyr Gly Val Pro Pro TyrLeu Leu Arg Tyr Pro Lys Gln Met 130 135 140 tgg gcc gag ggc tac acc ttccct ccc cct gcc atc cgc gac gag ttc 480 Trp Ala Glu Gly Tyr Thr Phe ProPro Pro Ala Ile Arg Asp Glu Phe 145 150 155 160 cgc cgc gtc tgg ccc gacgcc ggc gac gac tac cac cgc ttc tgc tcc 528 Arg Arg Val Trp Pro Asp AlaGly Asp Asp Tyr His Arg Phe Cys Ser 165 170 175 gcc atg gag gag tac gactcg tcg atg aga gct ctg ggc gag agg ctc 576 Ala Met Glu Glu Tyr Asp SerSer Met Arg Ala Leu Gly Glu Arg Leu 180 185 190 ctc gcc atg ttc ttc aaggcg ctc ggg ctc gcc ggc aac gat gcc ccc 624 Leu Ala Met Phe Phe Lys AlaLeu Gly Leu Ala Gly Asn Asp Ala Pro 195 200 205 ggc ggc gag acc gag cggaag atc cgc gaa acg ttg acg tcg tcg acg 672 Gly Gly Glu Thr Glu Arg LysIle Arg Glu Thr Leu Thr Ser Ser Thr 210 215 220 att cac ctc aac atg ttccct agg tgt cca gat cca gac cgg gtg gtc 720 Ile His Leu Asn Met Phe ProArg Cys Pro Asp Pro Asp Arg Val Val 225 230 235 240 ggg ctg gcg gcg cacacg gac tca ggc ttc ttc acc ttc atc ctg cag 768 Gly Leu Ala Ala His ThrAsp Ser Gly Phe Phe Thr Phe Ile Leu Gln 245 250 255 agc ccc gtg ccg gggttg cag ctg ctc cgc cac cgg ccg gac cgg tgg 816 Ser Pro Val Pro Gly LeuGln Leu Leu Arg His Arg Pro Asp Arg Trp 260 265 270 gtg acg gtt ccg gggacg ccg ggg gcg ctc atc gtc gtc gtc ggc gat 864 Val Thr Val Pro Gly ThrPro Gly Ala Leu Ile Val Val Val Gly Asp 275 280 285 ctc ttc cat gtg ctcacc aac ggg cgc ttc cac agc gtg ttc cac cgc 912 Leu Phe His Val Leu ThrAsn Gly Arg Phe His Ser Val Phe His Arg 290 295 300 gcc gtc gtg aac cgggag aga gac cgg atc tcc atg ccc tac ttc ctc 960 Ala Val Val Asn Arg GluArg Asp Arg Ile Ser Met Pro Tyr Phe Leu 305 310 315 320 ggt ccg ccg gccgac atg aag gtg aca cct ctc gtg gcg gcg ggg tcg 1008 Gly Pro Pro Ala AspMet Lys Val Thr Pro Leu Val Ala Ala Gly Ser 325 330 335 ccg gag agc aaggcc gtg tat cag gcc gtg aca tgg ccg gag tac atg 1056 Pro Glu Ser Lys AlaVal Tyr Gln Ala Val Thr Trp Pro Glu Tyr Met 340 345 350 gct gta agg gataag ttg ttc ggg aca aat ata tcg gcg ttg agc atg 1104 Ala Val Arg Asp LysLeu Phe Gly Thr Asn Ile Ser Ala Leu Ser Met 355 360 365 att cga gta gcgaag gaa gag gac aag gag agt tagaactatg gtatgattgc 1157 Ile Arg Val AlaLys Glu Glu Asp Lys Glu Ser 370 375 aattatccat gccagaaaaa aaaaaaaaaa1187 4 1122 DNA Oryza sativa CDS (1)..(1119) 4 atg ccg acg ccg tcg cacttg aag aac ccg ctc tgc ttc gac ttc cgg 48 Met Pro Thr Pro Ser His LeuLys Asn Pro Leu Cys Phe Asp Phe Arg 1 5 10 15 gcg gcg agg cgg gtg ccggag acg cac gcg tgg ccg ggg ctg gac gac 96 Ala Ala Arg Arg Val Pro GluThr His Ala Trp Pro Gly Leu Asp Asp 20 25 30 cac ccg gtg gtg gac ggc ggcggc ggc ggc ggc gag gac gcg gtg ccg 144 His Pro Val Val Asp Gly Gly GlyGly Gly Gly Glu Asp Ala Val Pro 35 40 45 gtg gtg gac gtc agg gcg ggc gacgcg gcg gcg cgg gtg gcg cgg gcg 192 Val Val Asp Val Arg Ala Gly Asp AlaAla Ala Arg Val Ala Arg Ala 50 55 60 gcg gag cag tgg ggc gcg ttc ctt ctggtc ggg cac ggc gtg ccg gcg 240 Ala Glu Gln Trp Gly Ala Phe Leu Leu ValGly His Gly Val Pro Ala 65 70 75 80 gcg ctg ctg tcg cgc gtc gag gag cgcgtc gcc cgc gtg ttc tcc ctg 288 Ala Leu Leu Ser Arg Val Glu Glu Arg ValAla Arg Val Phe Ser Leu 85 90 95 ccg gcg tcg gag aag atg cgc gcc gtc cgcggc ccc ggc gag ccc tgc 336 Pro Ala Ser Glu Lys Met Arg Ala Val Arg GlyPro Gly Glu Pro Cys 100 105 110 ggc tac ggc tcg ccg ccc atc tcc tcc ttcttc tcc aag ctc atg tgg 384 Gly Tyr Gly Ser Pro Pro Ile Ser Ser Phe PheSer Lys Leu Met Trp 115 120 125 tcc gag ggc tac acc ttc tcc cct tcc tccctc cgc tcc gag ctc cgc 432 Ser Glu Gly Tyr Thr Phe Ser Pro Ser Ser LeuArg Ser Glu Leu Arg 130 135 140 cgc ctc tgg ccc aag tcc ggc gac gac tacctc ctc ttc tgt gac gtg 480 Arg Leu Trp Pro Lys Ser Gly Asp Asp Tyr LeuLeu Phe Cys Asp Val 145 150 155 160 atg gag gag ttt cac aag gag atg cggcgg cta gcc gac gag ttg ctg 528 Met Glu Glu Phe His Lys Glu Met Arg ArgLeu Ala Asp Glu Leu Leu 165 170 175 agg ttg ttc ttg agg gcg ctg ggg ctcacc ggc gag gag gtc gcc gga 576 Arg Leu Phe Leu Arg Ala Leu Gly Leu ThrGly Glu Glu Val Ala Gly 180 185 190 gtc gag gcg gag agg agg atc ggc gagagg atg acg gcg acg gtg cac 624 Val Glu Ala Glu Arg Arg Ile Gly Glu ArgMet Thr Ala Thr Val His 195 200 205 ctc aac tgg tac ccg agg tgc ccg gagccg cgg cga gcg ctg ggg ctc 672 Leu Asn Trp Tyr Pro Arg Cys Pro Glu ProArg Arg Ala Leu Gly Leu 210 215 220 atc gcg cac acg gac tcg ggc ttc ttcacc ttc gtg ctc cag agc ctc 720 Ile Ala His Thr Asp Ser Gly Phe Phe ThrPhe Val Leu Gln Ser Leu 225 230 235 240 gtc ccg ggg ctg cag ctg ttc cgtcga ggg ccc gac cgg tgg gtg gcg 768 Val Pro Gly Leu Gln Leu Phe Arg ArgGly Pro Asp Arg Trp Val Ala 245 250 255 gtg ccg gcg gtg gcg ggg gcc ttcgtc gtc aac gtc ggc gac ctc ttc 816 Val Pro Ala Val Ala Gly Ala Phe ValVal Asn Val Gly Asp Leu Phe 260 265 270 cac atc ctc acc aac ggc cgc ttccac agc gtc tac cac cgc gcc gtc 864 His Ile Leu Thr Asn Gly Arg Phe HisSer Val Tyr His Arg Ala Val 275 280 285 gtg aac cgc gac cgc gac cgg gtctcg ctc ggc tac ttc ctc ggc ccg 912 Val Asn Arg Asp Arg Asp Arg Val SerLeu Gly Tyr Phe Leu Gly Pro 290 295 300 ccg ccg gac gcc gag gtg gcg ccgctg ccg gag gcc gtg ccg gcc ggc 960 Pro Pro Asp Ala Glu Val Ala Pro LeuPro Glu Ala Val Pro Ala Gly 305 310 315 320 cgg agc ccc gcc tac cgc gctgtc acg tgg ccg gag tac atg gcc gtc 1008 Arg Ser Pro Ala Tyr Arg Ala ValThr Trp Pro Glu Tyr Met Ala Val 325 330 335 cgc aag aag gcc ttc gcc accggc ggc tcc gcc ctc aag atg gtc tcc 1056 Arg Lys Lys Ala Phe Ala Thr GlyGly Ser Ala Leu Lys Met Val Ser 340 345 350 acc gac gcc gcc gcc gcc gccgac gaa cac gac gac gtc gcc gcc gcc 1104 Thr Asp Ala Ala Ala Ala Ala AspGlu His Asp Asp Val Ala Ala Ala 355 360 365 gcc gac gtc cac gca taa 1122Ala Asp Val His Ala 370 5 1948 DNA Oryza sativa genomic DNA sequence 5ctcgaggatc gaaaccaaaa ttaagggagc acaaaaaact atgacaaatg tttagttctg 60acaatgaact aaattagaac aaagcttgat ccgatcctat ccatttctga ttttgtgccg 120aacgatgcgg agagaagtta gttttttgta gataatgcaa gcccaaattt agccatgcta 180tctcgttatt aatcacgcga aagaaatggt catgccaaca aattaattta tcgtacatca 240ctagtcacag gcttttgtgc gttagccaac gagttcatgc agatcatgac atcgtcgtcg 300acctcgccga cctcgaccgc tggccgccgc cgcacacaat ggcgtcaccg ccgcctactt 360caacttccgc ggggcggagc gcgtgccgga gtcgcacgtg tggaagggga tgcacgagaa 420ggacaccgcg ccggtggcgg cggcggacgc ggacggcggc gacgcggtgc cggtggtgga 480catgagcggc ggcgacgacg ccgcggtggc ggcggtggcg cgcgcggcgg aggagtgggg 540cgggttcctg ctcgtcgggc acggcgtgac cgcggaggcc ctggcgcgcg tcgaggcgca 600ggcggcgcgg ctgttcgcgc tgccggcgga cgacaaggcg cgcggggcgc ggcggcccgg 660cggcgggaac accggctacg gcgtgccgcc gtacctcctc cggtacccga agcagatgtg 720ggccgagggc tacaccttcc ctccccctgc catccgcgac gagttccgcc gcgtctggcc 780cgacgccggc gacgactacc accgcttctg gtacgcgttt accgccgatc gatcgatcga 840tccgccattg cttgcatgca actaacctag ctagcttccg cgcgtgttcg tccgatccgg 900cccggccagc tccgccatgg aggagtacga ctcgtcgatg agagctctgg gcgagaggct 960cctcgccatg ttcttcaagg cgctcgggct cgccggcaac gatgcccccg gcggcgagac 1020cgagcggaag atccgcgaaa cgttgacgtc gtcgacgatt cacctcaaca tgtatgtaaa 1080ctcatatgga tgtggatttt ctatgcatag atgccatagc actgcaccca tcatttacat 1140acgattttga gaaaatataa gtttataaac aagctatatt taatctacaa ctaaaaaaac 1200aaaaataata aaatcaggca ataaatacta gtaaaatttg ttatttttac ttcgtgtgta 1260ggtcgaattt aattttacat atttatatag tgttttatac tattattgta atctatctta 1320tcaaattcta tgatttttta taactattta aactacatgt atgatacaca attagaaaat 1380acttttccat acaaatatat cttcacatgc aatggtgttt ggagctgatc gacacgtgtc 1440actctgacat ggccacacgc aggttcccta ggtgtccaga tccagaccgg gtggtcgggc 1500tggcggcgca cacggactca ggcttcttca ccttcatcct gcagagcccc gtgccggggt 1560tgcagctgct ccgccaccgg ccggaccggt gggtgacggt tccggggacg ccgggggcgc 1620tcatcgtcgt cgtcggcgat ctcttccatg tgctcaccaa cgggcgcttc cacagcgtgt 1680tccaccgcgc cgtcgtgaac cgggagagag accggatctc catgccctac ttcctcggtc 1740cgccggccga catgaaggtg acacctctcg tggcggcggg gtcgccggag agcaaggccg 1800tgtatcaggc cgtgacatgg ccggagtaca tggctgtaag ggataagttg ttcgggacaa 1860atatatcggc gttgagcatg attcgagtag cgaaggaaga ggacaaggag agttagaact 1920atggtatgat tgcaattatc catgccag 1948 6 2112 DNA Oryza sativa genomic DNAsequence 6 ttttttcctc tccaaatcta ttaattaatg atccatttca attcttcatcactgatttat 60 tcaccaatta attctctctt ttttttttct tccactacgc tccaaaacttctctccctat 120 atatacctct cccttgtact tgtccagttc ttacactcgt ctcactttactactcattcc 180 actattgtaa agtcatagaa aaaatttata tagagagaaa aaattagtgtttgttattgt 240 tactggcttt ctgccagacg agacgagcga gcgcgcgagt gtgttcctctctgagtcatc 300 tcgtcgtcgt cggcgatgcc gacgccgtcg cgcttgaaga acccgctctgcttcgacttc 360 cgggcggcga ggcgggtgcc ggagacgcac gcgtggccgg ggctggacgaccacccggtg 420 gtggacggcg gcggcggcgg cggcgaggac gcggtgccgg tggtggacgtcagggcgggc 480 gacgcggcgg cgcgggtggc gcgggcggcg gagcagtggg gcgcgttccttctggtcggg 540 cacggcgtgc cggcggcgct gctgtcgcgc gtcgaggagc gcgtcgcccgcgtgttctcc 600 ctgccggcgt cggagaagat gcgcgccgtc cgcggccccg gcgagccctgcggctacggc 660 tcgccgccca tctcctcctt cttctccaag ctcatgtggt ccgagggctacaccttctcc 720 ccttcctccc tccgctccga gctccgccgc ctctggccca agtccggcgacgactacctc 780 ctcttctggt atatatacat atatatatac tctcccatgc attccatgcacatacactct 840 acgtatatat ctacctctac gtatatatct acgtattgat ctacgtataatatacgcagt 900 gacgtgatgg aggagtttca caaggagatg cggcggctag ccgacgagttgctgaggttg 960 ttcttgaggg cgctggggct caccggcgag gaggtcgccg gagtcgaggcggagaggagg 1020 atcggcgaga ggatgacggc gacggtgcac ctcaactggt acccgaggtgcccggagccg 1080 cggcgagcgc tggggctcat cgcgcacacg gactcgggct tcttcaccttcgtgctccag 1140 agcctcgtcc cggggctgca gctgttccgt cgagggcccg accggtgggtggcggtgccg 1200 gcggtggcgg gggccttcgt cgtcaacgtc ggcgacctct tccacatcctcaccaacggc 1260 cgcttccaca gcgtctacca ccgcgccgtc gtgaaccgcg accgcgaccgggtctcgctc 1320 ggctacttcc tcggcccgcc gccggacgcc gaggtggcgc cgctgccggaggccgtgccg 1380 gccggccgga gccccgccta ccgcgctgtc acgtggccgg agtacatggccgtccgcaag 1440 aaggccttcg ccaccggcgg ctccgccctc aagatggtct ccaccgacgccgccgccgcc 1500 gccgacgaac acgacgacgt cgccgccgcc gccgacgtcc acgcataagctatagctact 1560 agctacctcg atctcacgca aaaaaaaaaa gaaacaatta atagagcaaaaaaaaaaaga 1620 aacaattaat agagcaaaaa aaaaaagaag agaaaatggt ggtacttgtgtttaaggttt 1680 cctccatgca aaatggtttg catgcatgca tgcaaagcta gcatctgcagctgcaagaat 1740 tacaagagca gagaagcaga cagctagatg gagataatta attaattaattaatctaatt 1800 aagcatgcaa taattaagat tattattctg atttcagaac tgaaaaaaaaagtgtggtta 1860 attaattatt ggttaggctt aattttatct agatgtagaa aaagaatcaagatcttcaag 1920 caagagagaa gaggatcgaa gaagaaggaa aagaaaacga aaaggacatgctgtgttgtc 1980 tcttctagtt gtaccctggc tgctgattaa gtgctttgtt ttgttgctgcaagcttgtcg 2040 ttactgatta ttagttagtt atgcatctaa ttgattaaac taatctgtttggcattttgg 2100 ctcgaggtcg ac 2112 7 20 DNA Artificial SequenceDescription of Artificial Sequencean artificially synthesized primersequence 7 gtngtnaarg tnggngarrt 20 8 20 DNA Artificial SequenceDescription of Artificial Sequencean artificially synthesized primersequence 8 ayytartcrt tggangtnac 20

1. A DNA encoding a protein having the gibberellin 3β-hydroxylaseactivity according to any one of the following (a) through (c): (a) aDNA encoding a protein comprising the amino acid sequence set forth inSEQ ID NO: 1 or 2, (b) a DNA comprising the coding region of thenucleotide sequence set forth in SEQ ID NO: 3 or
 4. (c) a DNA encoding aprotein comprising the amino acid sequence set forth in SEQ ID NO: 1 or2 in which one or more amino acids are substituted, deleted, added,and/or inserted.
 2. A DNA encoding an antisense RNA complementary to theDNA according to claim 1 or its transcription product.
 3. A DNA encodingan RNA having the ribozyme activity to specifically cleave thetranscription product of the DNA according to claim
 1. 4. A DNA encodingan RNA that suppresses the expression of endogenous DNA according toclaim 1 by co-suppression when the endogenous DNA is expressed in plantcells.
 5. A vector containing the DNA according to any one of claims 1through
 4. 6. A transformed plant cell harboring the DNA according toany one of claims 1 through 4 in an expressible state.
 7. A transgenicplant containing the transformed plant cell according to claim
 6. 8. Apropagative material of the transgenic plant according to claim
 7. 9. Aprotein encoded by the DNA according to claim
 1. 10. A method forproducing the protein according to claim 9, wherein said methodcomprises culturing the transformed cells carrying the DNA according toclaim 1 in an expressible state, and recovering the expressed proteinfrom said cells or the culture supernatant thereof.
 11. A method formodifying the plant growth, wherein said method comprises controllingthe expression level of the DNA according to claim 1 in plant cells. 12.A method for modifying a plant type, wherein said method comprisescontrolling the expression level of the DNA according to claim 1 inplant cells.